Antibiotics and anti-inflammatory drugs are two main categories which are
widely used in treatment of different diseases. That is why pharmaceutical
analysts are trying to develop different analytical methods for their
determination. The Pharmacopoeias are the main source to get the analysis
methods of most of the official drugs. Therefore Pharm. Eur. was selected to
get the official quantitative and qualitative method of analysis Some
representative drugs were chosen for the study in this thesis. Spiramycin,
Doxcyclin, Clindamycin and Roxithromycin were examples for antibiotics. For
anti-inflammatory drugs, Etodolac and Troxerutin were the selected examples.
According to the Pharm. Eur., HPLC and TLC techniques were the official
analytical methods for assay, identification and purity test for these drugs.
Through the literature of the official and nonofficial HPLC analysis of all
these drugs, conventional HPLC column was the only stationary phase which used
for their analysis. There are many disadvantages for the use of that type of
columns such as long analysis time. To the best of our knowledge there is no
reported UPLC-like method for any of these drugs. UPLC is a new type of fast
HPLC technique which characterised by the short analysis time. Therefore it
was important to transfer these official HPLC methods to UPLC-like method by
the use of new analytical columns which packed with more advanced stationary
phases such as monolithic and fused core columns. Moreover comparisons were
established among all selected stationary phases in terms of chromatographic
run time, different flow rates, the produced column back pressure, different
types of elution (gradient and isocratic), different mobile system
composition, and different sample solvents. Performance parameters as AF, Rs
and the theoretical plate number were also studied. The validation parameters
(linearity, precession, LOD, LOQ, robustness, reproducibility) were therefore
examined for the optimum conditions in each case. The aim of the work was to
get the most suitable chromatographic method which can be used instead of the
official HPLC method without affecting the resolution of peaks, sensitivity of
the method or all validation parameters. Very short analysis time was obtained
with all developed methods by using modern columns. For example, the official
chromatographic run time for the analysis of CLD was (12 min) and on its
transfer to UPLC-like method the analysis time was reduced to (2 min), for SPR
the run time reduced from official method (40 min) to developed method (5
min), while for ROX run time was very long with official method (100 min) but
by new developed method the run time was only (5 min). Validation parameters
were tested for all developed methods as Precision. The within-day
repeatability was in the range (0.52- 0.88%) with conventional column and
(0.41- 0.87%) with the modern columns. Furthermore, the between-days
repeatability was in the ra nge (0.66 –1.25%) with conventional and (0.50 –
0.96%) with modern columns. Modern columns gave more precise integration than
that of the conventional columns which can be attributed to the better peak
shape and reduced baseline noise. In fact, peak tailing in RP HPLC is
particularly prevalent when separating basic compounds. It causes a number of
problems including lower Rs, reduced sensitivity and poor precision and
quantitation. However symmetric peaks were obtained with the other used
stationary phases. The highest AF value was obtained with the use of
conventional stationary phase. For example, for Pharm. Eur. Analysis of ETD,
CLD or SPR, the obtained AF was 1.4, 1.4 and 1.5 respectively. However, with
the developed UPLC-like methods AF was reduced to (1.0 – 1.1) by using fused
core and monolithic columns respectively. In the official HPLC methods of some
of the selected drugs, the used temperature was not suitable for our lab work.
For example, in ROX or DOX official analysis, the temperature were 15 °C and
60°C respectively. However in the developed methods, an ambient temperature
was the optimum for the analysis. Moreover, ROX was efficiently separated from
its other components in the concentrated sample by the applying the developed
method. Acceptable resolution values were obtained with all developed methods.
ETD analysis as a representative example, poor separation of ETD from its
impurity CON was observed on applying the official method and the obtained Rs
was (Rs < 1). On the other hand, on applying the developed gradient elution
method, the Rs was raised to (Rs=2.5). Also, a developed UPLC-like method with
isocratic elution system for analysis of ETD and its impurity, the Rs values
were 1.4, 2.5 and 3.6 by using luna, monolithic and fused core columns
respectively. The developed methods were sensitive with low LOD and LOQ. For
example, the LOD and LOQ in the analysis of ETD by official method were 0.15
and 0.5 µg/ml respectively, while with the developed method LOD values were
reduced to 6, 3.8, 3.3 and 3 ng/ml was by using conventional, luna, monolithic
and fused core columns respectively. Also, the LOQ values were reduced to 20,
11.2, 14, and 10 ng/ml by using conventional, luna, monolithic and fused core
column respectively. In the analysis of CLD by the official method, the LOD
and LOQ values were 61 and 200 ng/ml respectively. However, on transferring to
UPLC-like method the LOD values were reduced to 13, 12 and 11 ng/ml by using
luna, monolithic and fused core columns respectively. LOQ values were reduced
as well to 47, 40 and 38 ng/ml by using luna, monolithic and fused core
columns respectively. The low LOD and LOQ values which obtained with the use
of monolithic and fused core columns can be attributed to the obtained low
background noise. The obtained column backpressure readings were watched. For
example, with CLD assay, the flow rate was 1ml/min with column lenght (50 mm),
the column backpressure readings were 16, 20, 74, 225 bar by using monolithic,
conventional, fused core and luna respectively. Generally higher backpressure
resulted with the use of luna because of the small particle size (2.5 µm dp).
On using fused core column the backpressure was reduced which attributed to
the new technology in particles (2.7 µm) with a high-capacity and very pure
porous silica layer which fused to a solid silica core. Monolithic column
showed the lowest backpressure reading due to the high permeability of its
bimodal structure. For some of the selected drugs “Etodolac, Spiramycin and
Troxerutin”, it was found that the official TLC methods were not able to get
efficient separation of the drug and its impurities spots. Therefore
developments of new TLC methods with the use of horizontal developing chamber
were studied. TLC mobile phase and the used sample solvent were optimized. In
TRX, The decrease in polarity of the sample solvent showed better results.
With the other drugs (SPR and ETD), phosphomolibdic acid reagent was used as
new derivatizing reagent with clear background of plate. That gave better
detection of the analyte spots rather than using UV lamp detection only which
was used in the official TLC method for ETD and anisaldhyde reagent for SPR.
In the official TLC purity test of Etodolac there are two different mobile
phases with two drying steps were used which time and cost are consuming. One
hour analysis time was consumed for the official method procedure. On the
other hand, in developed method, the analysis time was reduced to 3 and 1.5
min by using TLC and HPTLC plates respectively, with toluene and acetone in
the ratio 1:1 v/v as a mobile system. The developed methods were effective to
separate CON and ETD by using TLC and HPTLC plates. The measured TLC Rf values
0.54 and 0.60 for ETD and CON respectively. On the other hand, HPTLC Rf values
were 0.54 and 0.67 for ETD and CON respectively. No separation of components
of SPR sample was observed by applying official TLC method with 22 min
analysis time. However, seven different spots (which related to SI, II, III
and other four components in the same sample mixture) were observed by using
the developed method and the analysis time was reduced to 3 and 2 min by using
TLC and HPTLC plates respectively. According to DAB 1999 procedure, TRX was
analysed by TLC methods. Poor shaped spots were observed with Rf values 0.22
and 0.08 for the main spot and blue spot respectively which are less than
normal Rf range. That may be due to a poor resolution and separation of the
TRX components. By reducing the sample solvent polarity and increasing of the
polarity of mobile system, the Rf values and the shape of the separated spots
were improved. The obtained Rf values were 0.43 and 0.24 for the main spot and
blue spot respectively to be within the expected ranges of DAB, and the
developing time was 4 and 2 min by using TLC and HPTLC plates respectively.
However, the developed method with using concentrated sample was able to
separate the minor compound. The developed TLC method for analysis of SPR was
applied to preparative TLC. The developed bands were scratched, extracted and
analyzed with HPLC for comparison and confirmation. The obtained results were
matched. By using new developed HPLC method with isocratic elution system for
ETD analysis with different types of columns and the run time was 2 min. The
method was efficient to separate 0.03 µg of CON/ ml of ETD sample in a
concentration 0.003 mg/ml. However, the developed TLC and HPTLC methods were
also efficient to separate CON from ETD but in 0.05 mg of CON/ml of ETD sample
(1mg/ml) within 3 and 1.5 min developing times respectively. By using the
UPLC- like method for the analysis of SPR (0.25 mg/ml) at F= 0.8 ml/min, many
peaks were observed within 5 min run time. On the other hand, by applying the
developed TLC and HPTLC methods, seven different spots were developed with SPR
(4 mg/ml) within 3 and 2 min developing times respectively. In all examined
official HPLC and TLC methods a significant improvement could be obtained by
using UPLC-like methods and optimized conditions of HPTLC methods
respectively.